Method for determining the prognosis of a patient with a neurological disease

ABSTRACT

The present invention provides a method for the determining the prognosis for a patient diagnosed with a neurological disease. The present invention also provides a method for the identification of human subjects for placement in clinical drug trials of drugs being tested for the treatment of neurological disease and for determining a patient&#39;s future disease risk.

BACKGROUND OF THE INVENTION

[0001] The invention relates to a method for determining the prognosisof a patient with a neurological disease.

[0002] Neurological diseases include Alzheimer's disease (AD),Creutzfeldt-Jakob disease, Huntington's disease, Lewy body disease,Pick's disease, Parkinson's disease (PD), amyotrophic lateral sclerosis,multiple sclerosis (MS), neurofibromatosis, and diseases without anecessary genetic component such as brain injury, stroke andmulti-infarct dementia (MID). Most of these diseases are typified byonset during the middle adult years and lead to rapid degeneration ofspecific subsets of neurons within the neural system, ultimatelyresulting in premature death. There are no known cures and few therapiesthat slow the progression of these diseases.

[0003] Parkinson's disease (PD) is a common neurodegenerative disorderwhich first appears in mid- to late-life. Familial and sporadic casesoccur, although familial cases account for only 1-2 percent of theobserved cases. The neurological changes which cause this disease aresomewhat variable and not fully understood. The disorder generallydevelops asymmetrically with tremors in one hand or leg and progressesinto symmetrical loss of voluntary movement. Eventually, the patientbecomes incapacitated by rigidity and tremors. In the advanced stagesthe disease is frequently accompanied by dementia.

[0004] Diagnosis of both familial and sporadic cases of Parkinson'sdisease can only be made after the onset of the disease symptoms.Anticholinergic compounds, propranolol, primidone and levadopa arefrequently administered to modify neural transmissions and therebysuppress the symptoms of the disease, though there is no known therapywhich halts or slows the underlying progression.

[0005] Multiple Sclerosis (MS) is a neurodegenerative disease of thebrain and spinal cord in which a breakdown occurs in the myelinsheathing of the nerve fibers. MS is currently incurable and treatmentsare few and usually result in only temporary improvements of the diseasesymptoms.

[0006] Stroke is the sudden death of a portion of the brain cells due toa lack of oxygen. A stroke occurs when blood flow to the brain isimpaired resulting in abnormal brain function. Brain blood flow can beimpaired by blockage or rupture of an artery to the brain.

[0007] In the United States, about 400,000 people a year will sufferfrom a stroke, and up to 40% of these strokes may be fatal. The cost ofstrokes is not just measured in the billions of dollars lost in work,hospitalization, and the care of survivors in nursing homes. The majorcost of a stroke is the loss of independence that occurs in 30% of thesurvivors. What was a self-sustaining and enjoyable life style may losemost of it's quality after a stroke and family members can often findthemselves in a new role as caregivers.

[0008] Other cerebral vascular diseases that present similar sequelae tostroke are multi-infarct dementia (MID), vascular dementia (VaD), andcardiovascular injury or accident. In addition, diseases such as AIDScan often have vascular dementia as a complication. As with the abovediseases, there are no known cures for these diseases and most therapiesonly aid rehabilitation or lower the risk of having another vascularincident.

[0009] Apolipoprotein E (apoE) functions as a ligand in the process ofreceptor mediated internalization of lipid-rich lipoproteins. ApoE isprobably also involved in reverse lipid transport. In the centralnervous system (CNS), apoE plays a central role in the mobilization andredistribution of cholesterol and phospholipid during membraneremodeling associated with synaptic plasticity. The importance of apoEin the brain is further underscored by the absence of other key plasmaapolipoproteins such as apoA1 and apoB in the brain.

[0010] The apoE gene on chromosome 19 has three common alleles (E2, E3,E4), which encode three major apoE isoforms. The frequency of the apoE4allele has been shown to be markedly increased in sporadic Alzheimer'sDisease (AD) and late onset familial Alzheimer's disease (AD). This genedosage effect was observed in both sporadic and familial cases (i.e., asage of onset increases, E4 allele copy number decreases). Women, who aregenerally at a greater risk of developing Alzheimer's disease, showincreased apoE4 allele frequency when compared to age matched men.

[0011] The cholinergic hypothesis of geriatric memory dysfunction hasraised some fundamental questions regarding the heterogeneity ofresponses toward different cholinomimetics in AD. The absence of clearbeneficial effects of choline and lecithin on geriatric patients withand without AD is still perplexing. Furthermore, multiple clinicalstudies using esterases inhibitors such as physostigmine and tacrinehave shown that contrary to results found in young subjects, the optimalacute dose necessary to facilitate performance on memory tasks variedconsiderably among individual aged subjects.

[0012] Neurological diseases provide a unique series of complicationsfor the clinicians, patients, and care givers; the diseases oftenprogress rapidly and disrupt a vast number of major life functions. Theprogressive nature of these diseases makes the passage of time a crucialissue in the choice and administration of different treatment options.It would be desirable to know the severity of the prognosis for patientsdiagnosed with various neurological diseases.

SUMMARY OF THE INVENTION

[0013] We have discovered a method for determining the prognosis ofpatients with a non-AD neurological disease such as Parkinson's disease,Multiple Sclerosis, or stroke. Our prognostic methods provide aprognosis for the patient, including a prediction of the relativeoutcome of the patient in terms of rate of progression, severity ofdisease symptoms, and longevity. The prognostic methods allowclinicians, patients, and family members to make informed choices abouttherapeutic regimes. This method will also provide for more rapid andcost effective treatment by determining the relative appropriateness ofvarious therapeutic and palliative choices. Even where drug therapy isinappropriate, the prognostic method will provide patients, and theirfamily members, a more informed and realistic expectation of patientoutcome including an insight into the most effective rehabilitationstrategy, and a forecast of the patient's risk for future disease.

[0014] In the first aspect, the invention provides a method ofdetermining the prognosis for a patient diagnosed with a non-ADneurological disease. The method includes: a) identifying a patientalready diagnosed with a non-AD disease; b) determining the apoEgenotype or phenotype of a patient; and c) converting the data obtainedin step b) into a prognosis determination. The prognosis may include aprediction of drug efficacy, patient outcome, and patient risk forfuture disease events. In preferred embodiments, the method of theinvention may further include the steps of determining the BChE genotypeor phenotype of a patient, obtaining a patient profile, which may,preferably, include the patient's sex, age, and/or genotype (e.g.,presenilin, apolipoprotein E, or BChE genotype).

[0015] In other preferred embodiments of the prognostic method, thepatient is diagnosed with a disease selected from the group consistingof: Parkinson's disease (PD), multiple sclerosis (MS), and stroke whichshall also include multi-infarct dementia (MID), vascular dementia(VaD), and cardiovascular injury or accident, for example, as acomplication of AIDS.

[0016] In a second aspect, the invention provides a method fordetermining the prognosis of future risks of disease in a asymptomaticmammal. In preferred embodiments the mammal is a human and the methodfurther involves a determination of the mammals BChE genotype orphenotype, obtaining a patient profile, which may, preferably, includethe mammal's sex, age, and/or genotype (e.g., presenilin, apolipoproteinE, or BChE genotype).

[0017] In a related aspect, the invention provides a kit for performingthe prognosis. The kit includes a means for converting the patientprofile into a prognosis. In a preferred embodiment, the kit contains ameans for performing the steps of the conversion. In another preferredembodiment, the kit contains a means for compiling the data for saidpatient profile and for formatting said patient profile. In anotherpreferred embodiment, the kit contains a computer software program toperform the data analysis.

[0018] It should be noted that the therapies suggested by the prognosticmethod may be used alone, or in combination with other known therapiesthat are not otherwise contraindicated for the patient.

[0019] For the purpose of the present invention the following terms aredefined below.

[0020] “Non-AD Neurological disease” means any disease other thanAlzheimer's disease, which involves the neuronal cells of the nervoussystem. Specifically included are: prion diseases (e.g.,Creutzfeldt-Jakob disease); pathologies of the developing brain (e.g.,congenital defects in amino acid metabolism, such asargininosuccinicaciduria, cystathioninuria, histidinemia,homocystinuria, hyperammonemia, phenylketonuria, and tyrosinemia, andfragile X syndrome); pathologies of the mature brain (e.g.,neurofibromatosis, Huntington's disease, depression, amyotrophic lateralsclerosis, multiple sclerosis, and stroke); conditions that strike inadulthood (e.g. Creutzfeldt-Jakob disease, Huntington's disease, Lewybody disease, Parkinson's disease, Pick's disease, amyotrophic lateralsclerosis, multiple sclerosis, neurofibromatosis), brain injury, stroke,multi-infarct dementia (MID), vascular dementia (VaD), pathologies ofthe brain (e.g., brain mishaps, brain injury, coma, infections byvarious agents, and dietary deficiencies) and, cardiovascular injury oraccident, for example, as a complication of AIDS.

[0021] “Drug efficacy” means the a determination of an appropriate drug,drug dosage, administration schedule, and prediction of therapeuticutility.

[0022] “Already diagnosed” means already diagnosed as having aneurological disease or having a genetic predisposition or risk foracquiring a neurological disease.

[0023] “Patient profile” means data pertaining to the patient for whomthe prognostic analysis is being performed. Data may include informationon the patient's diagnosis, age, sex, and genotype. The patient'sprofile may also include materials from the patient such as blood, serumprotein samples, cerebrospinal fluid, or purified RNA or DNA.

[0024] “Asymptomatic” means that the mammal or human subject has noclinical symptoms of a disease but nonetheless may be a “silent” carrierof a genotype determined by the method of the invention to result lateror be likely to result later in the onset of disease symptoms.

[0025] “ApoE genotyping” means determination of the type and number ofapoE alleles present in the patient, whether determined by nucleic acidsequencing, PCR or RT-PCR amplification, examination of apoE protein, orby other methods available to those skilled in the art.

[0026] “Allele load” means the relative ratio of apoE2, 3, and 4 allelesin the patient's chromosomal DNA. The allele load may be determined bycomparing the relative numbers of the patient's already known apoEallele types.

[0027] “PCR or RT-PCR amplification” means subjecting a DNA sample to aPolymerase Chain Reaction step or an RN-A sample to a ReverseTranscriptase-Polymerase Chain Reaction step, such that, in the presenceof appropriately designed primers, a DNA fragment is synthesized orfails to be synthesized and thereby reveals the allele status of apatient.

[0028] “BChE genotype” means a determination of the patient's allelesthat encode the butyrylcholinesterase gene product. This may beaccomplished by nucleic acid sequencing, RT-PCR, PCR, examination of theBChE protein, a determination of the BChE enzyme activity, or by othermethods available to those skilled in the art.

[0029] “BCHE-K allele” means the polymorphism of thebutyrylcholinesterase (BCHE) gene which has a point mutation atnucleotide 1615 that changes amino acid residue 539 from alanine tothreonine and can result in an enzyme with reduced catalytic activity.Other polymorphisms of this locus exist (e.g., deletions (BCHBE*FS4),missense mutations (BCHE*24 M, * 1005, *250P, *267R, *330I, *365R,*418S, *515C, *539T), and nonsense mutations (BCHE*119STOP, *465STOP))and are included within the scope of the invention.

[0030] “Prognosis” means a method whereby diagnostic data, including thepatient's neurological diagnosis and genetic data, such as the patient'sapoE and BChE genotype, are processed to provide therapeutic options andprognoses. Processing may include, but not be limited to, the collectionof a patients medical history including age and sex, apoE and BChEgenotyping using appropriately designed primers and using a RT-PCR orPCR amplification step, apoE and BChE phenotyping using anantibody-mediated method or enzymatic test, and statistical analysisstep that converts this raw data into a prognosis. The prognosis mayinclude a prediction of a patient's response to drug therapy, recoverytime, age of disease onset, treatment efficacy, rehabilitation time,symptomology of attacks, and risk for future disease. For example, ahigh apoE4 allele load could be used as a positive predictor for strokepatients that respond well under drug therapy and as negative predictorof PD and MS patient response to drug therapy. A prognosis may also bedetermined for asymptomatic and healthy subjects in order to forecastfuture disease risks an determine what preventive therapies should beconsidered or invoked in order to lessen these disease risks. Theprognosis may include the use of a computer software program to analyzepatient data and run statistical cross-checks against relationaldatabases that are constantly being updated.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Here we show that a correlation of age, sex, apoE genotype, andBChE genotype, may be used to formulate a prognosis for a given patientwith a neurological disease. The prognosis can include a prediction ofboth relative age of onset, rate of disease progression, and risk forfuture disease.

[0032] We have investigated the relationship between the apoE4 genotypeand cholinergic deficits, and we observed that the greater the number ofapoE4 alleles the lower the apoE level. Furthermore, reduction in ChATactivity in the hippocampus and temporal cortex of AD cases is inverselyproportional to the apoE4 allele copy number (i.e. where the apoE4allele copy number is increased the ChAT activity is decreased). Inaddition, we found that another presynaptic marker of cholinergicprojection, the nicotinic receptor, was markedly reduced in apoE4 ADsubjects. Conversely, we have found that a typical post-synaptic marker,M1-muscarinic receptor, is unaltered in AD versus non-AD subjects,irrespective of whether apoE4 is present or not. The M2-muscarinicreceptor, a composite pre- and post-synaptic marker, is also unaffectedby the apoE4 allele gene dosage. We have also observed that the presenceof the apoE4 allele lowers the age of onset of neurological disease andworsens the prognosis.

[0033] The above findings clearly indicate the existence of distinctgenetic entities in neurological disease which correlate withdifferential degrees of alterations of cholinergic innervation. In turn,the innervation level correlates with the prognosis, including theability to respond to cholinomimetic drugs.

[0034] We believe the correlation between apoE4 allele load andreductions in ChAT activity and nicotinic receptors may be explained byat least two distinct phenomena. First, phospholipids suchphosphatidylcholine (PC) and phosphatidylethanolamine (PE), that canserve precursors to choline in the synthesis of acetylcholine (Ach),could be transported into neurons via the classical apoE-LDL receptorpathway. An isoform-dependent impaired regulation of the transport ofphospholipids in the brain of apoE4 carriers could explain the reducedlevels of PC, PE and choline reported in AD (Pettegrew J. W., 1989, Ann.NY Acad. Sci., 568:5-28; Nitch R M et al., 1992, Proc. Natl. Acad. Sci.,89:1671-1675). This, in turn, may lead to decreased Ach syntheticcapacities. This hypothesis is consistent with membrane defects reportedin AD subjects such as changes in membrane fluidity in the hippocampusand in the platelets of AD patients. The loss of cholesterol reported inAD and the effect of apoE4 on nicotinic binding activity are consistentwith the apoE4/impaired lipid homeostasis hypothesis.

[0035] In addition to the above, the reduction in neuronal ChATactivities and choline levels in both AD and non-AD patients couldparallel the loss of cholinergic neurons. The analysis of the number ofacetylcholinesterase-positive neurons in the nucleus basalis of Meynert(NBM) and the diagonal band of Broca (DBB) in AD patients revealedmarked losses of cholinergic neurons in apoE4 carriers versus apoE3homozygous AD cases.

[0036] Although these observations were initially made in AD patients,we have discovered that our observations regarding apoE allele load anddrug therapies can be generalized to non-AD neurological diseasesbecause the underlying mechanism altered by the apoE allele load is notAD-specific. Our discovery indicates that the apoE4 allele load, takentogether with patient profile parameters, can predict individualvariations in a patients response to drug treatment, rehabilitation, andrisk for future disease. The method of the invention provides usefulpredictions for patients with diseases such as stroke, Parkinson'sdisease, and Multiple Sclerosis as discussed in the Examples herein.Prospective-retrospective analyses of patients that are either good orpoor responders to drugs designed to ameliorate the conditions ofstroke, Parkinson's disease, and Multiple Sclerosis, (e.g., aspirin,antithrombotics, ticlopidine (Ticlid™), levodopa-carbidopa, (Sinernet™)and interferon β-1B (Betaseron™)) are presented in the Examples. Wefurther propose that the method of the invention also can be used togenerate prognostic protocols for other cerebral vascular injuries thatinvolve similar disease mechanisms to stroke. Such diseases wouldinclude multi-infarct dementia (MID), cardiovascular injury, braininjury, or cerebral vascular pathologies as a complication AIDS.

[0037] The prognostics method is useful for allowing the physician toselect the most appropriate drug, drug dosage, administration orpalliative therapy for a patient. The invention also provides a methodfor forecasting future patient disease risk. This forecast method allowsthe patient and clinician to consider and invoke preventive therapeuticregimens before disease strikes or to at least lower the risk of futuredisease events.

[0038] The present invention will be more readily understood byreferring to the following examples which are given to illustrate theinvention rather than to limit its scope.

EXAMPLE I

[0039] Methods and Study Design

[0040] Determination of apoE Levels and Allele Load

[0041] Genotype analysis for each patient was performed using highmolecular weight DNA, or alternatively RNA, isolated from 5 mls of wholeblood drawn from each patient. The apoE genotype was determined using anallele-specific primer extension method. Primers labeled D, E, F, G, andH were synthesized by Genosys Biotech (The Woodland, Tex.) using primersequences provided in Main et al. (Main R. F. et al., 1991, J. Lipid.Res., 32:183-187). Reactions were carried out in a volume of 50 uLcontaining 1 ug of DNA; deoxyadenosine triphosphate, deoxycytidinetriphosphate, deoxythymidine triphosphate and deoxyguanosinetriphosphate, each 0.2 mmol/L; 10% dimethyl sulfoxide; 12.5 pmol ofeither primer D, E, F, or G; 25 pmol of primer H; and 10 uL of 10× PCRreaction buffer (Vector Biosystem, Toronto, ONT.). The DNA in thereaction mixture was first denatured for 10 min. at 96° C. and thencooled to 4° C. One unit of Taq polymerase (Vector Biosystem, Toronto,ONT.) was then added to each sample. Each sample was reheated for 2 min.at 96° C. and subjected to 30 cycles in a thermal cycler with each cycleconsisting of a 10 sec denaturation at 96° C., 30 sec annealing at 58°C., and 1 min. extension at 65° C. The reaction products were visualizedby electrophoresis of 10 uL of the reaction mixture in a 1% agarose gelcontaining TPE buffer (0.08 mol/L Tris-phosphate, 0.002 mol/L EDTA,Sigma, St-Louis, USA) and ethidium bromide (0.15 ug/mL) for 1 hr at 67v.The gels were then photographed and the banding profile was compared toknown standards.

[0042] Alternatively, the apoE phenotype can be determined in a patientusing a serum or cerebrospinal fluid sample. Proteins are size separatedon a 25 cm SDS polvacrylamide gel (10%) and transferred onto anitrocellulose filter using a BIORAD™ Trans-blot cell and detection ofthe apoE protein is performed using a polyclonal antibody raised againsthuman apoE protein (International Immunology Corp., Calif., Dil.1:2000). To control for antibody specificity, adsorption of theanti-apoE antibody with purified human apoE protein (MW 34-36 kDa) isperformed see if will specifically block apoE detection. Molecularweight markers (Rainbow markers, Amersham) are run in adjacent wellswhile visualization of the bands is done with a chemiluminescencedetection kit (Amersham, Cat. No. RPN 2100). Quantification of theautoradiographic signals is performed using a MCID image analysis system(Ste-Catherine, Ontario) equipped with the 1D-gel analysis software.

[0043] Patients who completed the drug trial were selected for adetermination of their apoE genotype as previously described (above andPoirier et al., 1993, Lancet 342:697-699). Patients were examined forthe impact different apoE genotypes (e.g., 2/2, 3/2, 3/3, 3/4, 2/4 and4/4) had on a patient's response to drug therapy, recovery time, age ofdisease onset, treatment efficacy, rehabilitation time, symptomology ofattacks, and risk for future disease.

EXAMPLE II Relationship Between ApoE Genotype and Drug Therapy Outcomein Patients Suffering from Stroke

[0044] We have analyzed 51 patients suffering from stroke to determineif there is a relationship between apoE genotype and drug therapyoutcome. Our small study group was composed of Caucasian females,currently diagnosed as suffering from stroke, and currently undertreatment with either aspirin or anti-thrombotic drugs (e.g. Ticlid™).Although our study group is small, and is not randomized by sex, thepatients in this study have a apoE allele distribution similar to a muchlarger randomized North American-population (see Table 1). To determinea patients apoE genotype, 5 mls of whole blood was drawn from eachpatient and used as a source of genetic material for apoE alleledetermination as described in Example I. The patient's apoE genotype wasthen compared with information from the patient's medical file. We havemeasured the speed at which patients recover from a stoke incident andthe duration of their rehabilitation, in order to assess the potentialrelationship between a patient's apoE genotype and stroke prognosis.

[0045] Results TABLE 1 apoE Allele Distribution of Study Group Comparedto Representative Population % Population Genotype (Canada) Study GroupE4/E4 3.9% 0% E4/E3 20.6% 27% E4/E2 9.8% 2% E3/E3 61.8% 59% E3/E2 2.0%12% E2/E2 2.0% 0%

[0046] In order to determine the relationship between apoE genotype andthe time it takes a patient to make a complete recovery from a strokeincident, we have analyzed the data as follows. We have divided thestudy group into two populations, those patients which recovery quicklyon drug therapy (fast responders) and those patients that recoveryslowly on drug therapy (slow responders) and asked what is the apoEallele representation (Table 2). TABLE 2 apoE Genotype Distribution inStroke Patients that Make a Complete Recovery Under Drug Therapy Quicklyvs. Slowly % population This Slow Fast Genotype (Canada) Study ResponderResponder E4/E4 3.9% 0% 0% 0% E4/E3 20.6% 27% 20% → 47% E4/E2 9.8% 2% 3%0% E3/E3 61.8% 59% 63% ← 47% E3/E2 2.0% 12% 14% ← 7% E2/E2 2.0% 0% 0% 0%

[0047] When the genotypes of these two populations were compared, weobserved a link between the speed of recovery from stroke and being acarrier of the apoE4 allele. The fastest responders to drug therapyfollowing stroke, were patients carrying the apoE4 allele (Table 2).When these data were analyzed as a function of apoE4 allele load,patients with a greater apoE4 allele load were over represented asmaking a good recovery (Table 3). TABLE 3 Comparison of apoE4 GenotypeLoad and Stroke Recovery Genotype poor good +E4 19% → 32% −E4 81% ← 68%

[0048] In order to determine the relationship between apoE genotype andage of disease onset, apoE genotype data was analyzed as a function ofthe age of the patient when the first stroke incident occurred (Table4). In this study, the majority of the patients, 74%, were non-apoE4carriers, and the genotypes were well distributed into all age groups,which minimized any favored results due to longevity possibly beinglinked to apoE genotype. TABLE 4 apoE4 Allele Distribution as a Functionof Age When Patient Suffered Stroke Incident Genotype 50-60 70 80 90E4/E4 0.0% 0.0% 0.0% 0.0% E4/E3 22.2% 20.0% 30.8% 66.7% E4/E2 11.1% 0.0%0.0% 0.0% E3/E3 66.7% 73.3% 53.8% 16.7% E3/E2 0.0% 6.7% 15.4% 16.7%E2/E2 0.0% 0.0% 0.0% 0.0%

[0049] In addition, analysis of this data by age group does not showmajor differences regarding treatment efficacy, even though, patients inthe 70 year old group appear to have the best overall response to drugtreatment (Table 5). TABLE 5 Drug Treatment Efficacy for Stroke Patientsas a Function of Age % Study 50-60 70 80 90 Poor 100% 15% 31% 31%  8%Average 100% 18% 18% 36% 14% Good 100% 23% 46%  8% 15% Very good 100% 0% 50%  0%  0%

[0050] In order to determine if the apoE genotype influences the totaltime a stroke patient requires for rehabilitation (a long rehabilitationperiod after a stroke incident is common), we have analyzed patientrehabilitation time as a function of the patient's apoE genotype. Weobserved a direct and positive relationship between the presence of anapoE4 allele and a short rehabilitation time (Table 6). TABLE 6 TotalRehabilitation Time Required after Stroke Incident as a Function ofPatient apoE Genotype % Population Genotype (Canada) % Study Quick SlowE4 15.20% 21% 28% → 19% E3   77% 69% 64% ← 74% E2  7.80% 10% 8% 7%

[0051] In order to assess whether a patient's apoE genotype influencesrecovery directly after a stroke attack, we have analyzed a patient'sability to make an immediate recovery following stroke as a function ofthe patient's apoE genotype. We observed that the speed of recoveryimmediately following a stroke attack is not apoE genotype dependent(Table 7). TABLE 7 Patient Recovery Ability Immediately Following aStroke as a Function of Patient apoE Genotype % population Genotype(Canada) % Study Quick Slow +E4 15.2% 21% 21% 25% −E4 84.8% 79% 79% 75%

[0052] In summary, we have discovered that a direct link exists betweenthe presence of an apoE4 allele and a stroke patient's outcome. Webelieve that the apoE4 allele may be beneficial to the stroke patient.We further predict, that the ability to upregulate the expression orstability of the apoE4 gene product, may have beneficial consequencesfor a patient suffering from stroke. This invention proposes that theability of a drug to increase apoE4 gene expression or stability wouldimplicate that drug as a therapeutically-effective drug for patientswith stroke.

EXAMPLE III Relationship Between ApoE Genotypes and Drug Therapy Outcomein Patients Suffering from Parkinson's Disease

[0053] We have analyzed 59 patients suffering from Parkinson's disease(PD) to determine if there is a relationship between a patient's apoEgenotype and drug therapy outcome. Our small group was composed ofCaucasian males, diagnosed as suffering from PD, and currently undertreatment with levadopa-carbidopa (Sinernet™). These patients were notsuffering from any other central nervous system disease. To determinethe patient's apoE genotypes, 5 mls of whole blood was drawn from eachpatient and used as a source of genetic material for apoE alleledetermination as described in Example I. The patient's apoE genotype wasthen compared with information from the patient's medical file. Ouranalysis was based on comparing patient response to a drug treatmentdesigned to improve symptoms of tremor and rigidity, as a function ofthe patient's apoE genotype.

[0054] Results TABLE 8 ApoE Genotype Distribution of Study Group, aLarger Population, and Across Different Age Groups % population Genotype(Canada) % Study 40-50 60 70 80 E4 15.20% 16.3% 18.8% 10.7% 20.0%  0.0%E3 77% 68.8% 68.8% 71.4% 70.0% 100.0% E2  7.80% 15.0% 12.5% 17.9% 10.0% 0.0%

[0055] Although our study group is small, and is not randomized by sex,the patients in this study have an apoE allele distribution similar to amuch larger randomized North American-population (Table 8). We alsoobserved a similar apoE allele distribution among different age groups,suggesting that apoE genotype is not linked to longevity (Table 8). Wecompared the average age of patients who responded well to drug therapy(good responder) versus those patients that did poorly (bad responder),and observed a similar average age (64-67). This result furtherconfirmed the lack of an age component in this data set. Furthermore,analysis of the apoE allele load between good and bad responders to drugtherapy revealed that age differences did not influence treatmentoutcome.

[0056] In contrast, we observed a strong negative correlation betweenpatient response to drug therapy and apoE4 allele load. Patients with noapoE4 allele showed a better response, as measured by improvements insymptoms of rigidity and tremor, then did those patient's with an apoE4allele (Table 9). Thus, we conclude that a negative correlation forpatients carrying an apoE4 allele and treatment outcome for Parkinson'sDisease. TABLE 9 Drug Response of Parkinson's Disease Patients withDifferent apoE Genotypes % population Bad Good Genotype (Canada) % Studyresponder responder E4/E4 3.9% 0% 0% 0% E4/E3 20.6% 17% 20% ← 8% E4/E29.8% 5% 4% 4% E3/E3 61.8% 63% 68% → 72% E3/E2 2.0% 14% 4% → 16% E2/E22.0% 2% 4% 0% +E4 15.2% 16% 18% ← 9% −E4 84.8% 84% 81% → 91%

EXAMPLE IV Relationship Between ApoE Genotype and Drug Therapy Outcomein Patients with Multiple Sclerosis

[0057] We have analyzed 65 patients suffering from multiple sclerosis(MS) to determine if there is a relationship between the patient's apoEgenotype and drug therapy outcome. Our small group was composed ofCaucasian females, diagnosed as suffering from MS, and currently undertreatment with interferon β-1B (Betaseron™). To determine a patient'sapoE genotype, 5 mls of whole blood was drawn from each patient and usedas a source of genetic material for apoE allele determination aspreviously described (see Example I). The patient's apoE genotype wasthen compared with information from the patient's medical file. Ouranalysis was based on comparing a patient's response to drug treatmentfor symptomatic MS exacerbations as a function of the patient's apoEgenotype.

[0058] Results

[0059] We analyzed the frequencies of apoE genotypes in our study groupas compared to a larger representative population. We observed that thepatients in this study had an apoE allele distribution similar to a muchlarger randomized North American-population (Table 10). We also observeda similar distribution among different age groups suggesting that theapoE genotype is not linked to longevity (Table 10). TABLE 10 ApoEGenotype Distribution of Study Group, Larger Population, and AmongstDifferent Age Groups % population Genotype (Canada) % study <3030-40 >40 E4 15.20% 15.6%  9.7% 21.6% 12.5% E3 77% 66.7% 67.7% 62.2%68.8% E2  7.80% 17.8% 22.6% 16.2% 18.8%

[0060] In order to determine the relationship between a patient's apoEgenotype and a patient's response to drug therapy designed to lessen thesymptomatic flare-ups of MS, we have analyzed the data as follows. Wehave divided the study group into two populations representing thosepatients which have fewer attacks on drug therapy (good responders) andthose patients that have many attacks while on drug therapy (badresponders) and asked what is the apoE allele representation (Table 11).We have observed that non-apoE4 patients responded better to drugtherapy than did those patients that carried an apoE4 (Table 11). TABLE11 ApoE Genotype Distribution in MS Patients Responding Well to DrugTherapy Versus Those Patients Responding Poorly % population % Bad GoodGenotype (Canada) study responders responders E4/E4 3.9% 0.0% 0.0% 0.0%E4/E3 20.6% 15.4% 8.3% 8.3% E4/E2 9.8% 6.2% 6.9% ← 1.7% E3/E3 61.8%60.0% 23.3% → 33.3% E3/E2 2.0% 16.9% 5.0% → 13.3% E2/E2 2.0% 1.5% 1.7%0.0%

[0061] When we analyzed these MS patients by apoE4 allele presence, thecorrelation between those patients that respond well and the lack of anapoE4 allele is striking (Table 12). Lack of an apoE4 allele is clearlypredictive of a good response to drug therapy. TABLE 12 ApoE4 AlleleLoad in MS Patients Responding Well to Drug Therapy Versus ThosePatients Responding Poorly % population Genotype (Canada) % study BadGood +E4 15.2% 16.5% 6.7% ← 5.6% −E4 84.8% 65.9% 20.0% → 43.3%

[0062] In another analysis of MS patients, we studied the correlationbetween a patient's apoE4 allele load and the qualitative nature of thepatient's MS attacks each year while undergoing drug treatment. Thefollowing results are subdivided into 8 groups, representing patient'swith mild attacks reacting badly to treatment compared with patientswith mild attacks having a good response to treatment, and patients withsevere attacks compared in the same manner (Table 13). We observed thatan apoE4 allele load had a negative impact on the efficacy of drugtreatment for MS patients as measured by a diminution in the number ofworsening attacks (Table 13). Thus, we conclude that an apoE4 alleleload is a strong predictor of poor MS patient response to drugtreatment. TABLE 13 ApoE4 Allele Load in MS Patients with a QualitativeChange in Attacks Genotype mild-bad mild-good severe-bad severe-good +E43% 2% 5% 7% −E4 15% → 34% 14% → 20%

[0063] We believe these results demonstrate that the Apolipoprotein Egene is not only linked to the drug efficacy in Alzheimer's disease, butalso directly related to drug efficacy in different CNS diseases such asstroke, Parkinson's disease, and Multiple Sclerosis. Due to the specificrole of the apoE gene and its proposed role in cell regeneration andplasticity requirements specific to a disease, we predict that thedisease relevant genotype will vary from disease to disease.

EXAMPLE V Determination of Risk Prognosis and Therapeutic TreatmentOutcome of Stroke Events Using Genetic Analysis of apoE4 and BCHE-K

[0064] Stroke is an acute neurologic event leading to death of neuraltissue of the brain and resulting in loss of motor, sensory, and/orcognitive function. It is said to be the third leading cause of death inthe United States. Genetic predisposition may be important in thepathogenesis of stroke. Such predisposition may not only include genescontributing to elevated blood pressure but also genes actingindependently of blood pressure. Twins studies and familial aggregationsupport evidence for genetic factors contributing to stroke with apolygenic aetiology.

[0065] Genetic factors contributing to stroke pathogenesis are poorlyestablished. The following genes and polymorphisms have been implicatedin stroke: ACE insertion/deletion polymorphisms, factor V gene, factorVII gene, PIA2 polymorphism of the glycoprotein IIIa gene, ApoE, and theinterleukin 1β converting enzyme (ICE) gene family.

[0066] Butyrylcholinesterase (BChE) is expressed in most human tissues,but its precise metabolic function in the body is still unknown. Thepolymorphic gene variant BCHE-K, consisting of a point mutation atnucleotide 1615 (GCA to ACA) which chances alanine 539 to threonine, hasreduced catalytic activity (Bartels et al., Am. J. Hum. Genet.50:1086-1103, 1992). Recent research from Lehmann et al. (Hum. Mol.Genet. 11:1933-1936 (1997)) suggests that BCHE-K is associated with afurther increase in the risk of late-onset AD in apoE4 carriers.

[0067] We have discovered that the combination of apoE4 and BCHE-Kcontribute to define an individual's risk for the development of stroke.We have determined the ApoE4 and BCHE-K genotype for 50 female strokepatients and 64 age and sex matched healthy controls (Table 16). In thecontrol group, we observed 17 out of 64 subjects were heterozygous forthe BCHE-K allele, and 4 out of 64 subjects were homozygous carriers ofthe BCHE-K allele. We observed that 15 out of 64 subjects carried onecopy of the apoE4 allele. Both of these allele distributions fit aHardy-Weinberg equilibrium for the identified allele frequency (Table14). In the stroke patients group, 22 out of 50 subjects carried onecopy of BCHE-K, and 3 out of 50 subjects were observed to carry twoBCHE-K alleles. Of these 50 subjects, 15 were carriers of one apoE4allele. The identified carrier status was in agreement with aHardy-Weinberg population for the observed allele frequency (Table 14).TABLE 14 Allelic Frequencies of apoE4 and BCHE-K No. of subjects F:Mratio mean age AF BCHE-K AF ApoE4 controls 64 F only 72 0.195 0.117 >58years Stroke 50 F only 0.28 0.15 cases p = (X- 0.18 (NS) 0.6 (NS)square, Yates corr.)

[0068] For subjects 58 years of age and older, the allelic frequency ofBCHE-K was 0.195 in controls and 0.28 in the 50 stroke cases, providingan odds ratio of stroke of 1.6 (based on allele frequencies) and 2.1(based on carrier frequencies) (Table 17.). In ApoE4 carriers, the oddsratio of stroke was about 1 (Table 15). TABLE 15 Odds Ratios of Strokefor BCHE-K alleles Odds Odds ratio ratio subjects controls cases(alleles) 95% Cl (carriers) 95% Cl all 64 50 1.6 0.9-2.9 2.1 1.0-4.4ApoE4 carriers 15 15 1.3 0.3-5.6

[0069] The allelic frequency of apoE4 was 0.12 in controls and 0.15 in50 stroke cases, giving an odds ratio of stroke of 1.3 (based on allelefrequencies) and 1.4 (based on carrier frequencies) (Table 16). InBCHE-K carriers, the odds ratio of stroke was 1.9 (calculated forcarrier status) (Table 16). TABLE 16 Odds Ratios of Stroke for apoE4alleles Odds Odds ratio ratio subjects controls cases (alleles) 95% Cl(carriers) 95% Cl all 64 50 1.3 0.6-2.8 1.4 0.6-3.2 BCHE-K 21 25 1.90.5-7.2 carriers

[0070] Taking account of the carrier status of both gene mutations, wehave discovered that there is a two fold increase (8% vs 18%) in strokecases, compared to controls, in carriers with both the apoE4 and BCHE-Kallele (Table 17). This trend is also seen in the apoE4 carriers. TABLE17 Proportion of Control and Stroke Subjects with both BCHE-K and apoE4Alleles subjects Controls Stroke cases P = (X-square, Yates corr.) all5/64 (8%) 9/50 (18%) 0.17 (NS) ApoE4 carriers 5/15 (33%) 9/15 (60%) 0.27(NS)

[0071] In Table 18, we provide the odds ratio of stroke for subjectscarrying at least one allele of apoE4 and BCHE-K as compared to controlsubjects who have neither allele. In female subjects over 58 years ofage who carry both the apoE4 and BCHE-K alleles, the odds ratio ofsustaining a stroke was 2.8 fold higher than age matched controls. Thesedata predict for female carriers of this genetic status, an almost 3fold higher risk for stroke. TABLE 18 Odds Ratio of Stroke Odds ratioApoE4 BCHE-K controls Stroke (carriers) 95% Cl no no 33 21 Reference yesyes  5  9 2.8 0.9-9.2

[0072] In summary, we have discovered that determining an individuals'sapoE4 and BCHE-K allele status is a useful tool in the prediction of anindividual's risk for stroke. Furthermore, our results demonstrate thatprognostic forecasting could allow patients to start prophylactictherapies before disease strikes. For example, the risk of stroke couldbe calculated for asymptomatic and healthy individuals as young adultsand well before a stroke incident has occurred. Then as the individualages, preventive therapies could be invoked in order to prevent orlessen the likelihood of a catastrophic stroke incident later in life.

OTHER EMBODIMENTS

[0073] While the invention been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of theappended claims.

[0074] Other embodiments are within the claims.

What is claimed is:
 1. A method of determining the prognosis for apatient already diagnosed with a non-AD neurological disease, saidmethod comprising: a) identifying a patient with said disease; b)determining the apoE genotype or phenotype of said patient; c)converting the data obtained in step b) into a prognosis for saidpatient.
 2. The method of claim 1, wherein said prognosis includes aprediction of drug efficacy, patient outcome, or a forecast of patientdisease risk.
 3. The method of claim 1, wherein the method furthercomprises determining the BChE genotype or phenotype of said patient. 4.The method of claim 1, wherein said method further comprises obtaining apatient profile.
 5. The method of claim 4, wherein said patient profileincludes a determination of said patient's sex.
 6. The method of claim4, wherein said patient profile includes the genotype of said patient.7. The method of claim 4, wherein said patient profile includes the ageof said patient.
 8. The method of claim 1, wherein said patient isdiagnosed with a disease or determined as being predisposed to a diseaseselected from the group consisting of stroke, Parkinson's disease,multiple sclerosis, multi-infarct dementia (MID), vascular dementia,cardiovascular injury, and cardiovascular accident.
 9. The method ofclaim 1, wherein said patient is diagnosed as having stroke or as beingpredisposed to sustain a stroke.
 10. The method of claim 1, wherein saidpatient is diagnosed as having Parkinson's disease or as beingpredisposed to acquire Parkinson's disease.
 11. The method of claim 1,wherein said patient is diagnosed as having multiple sclerosis or asbeing predisposed to acquire multiple sclerosis.
 12. A method foridentifying a patient for participation in a clinical trail of a drugfor the treatment of a neurological disease, said method compromising:a) identifying a patient already diagnosed with said disease or as beingpredisposed to acquire or be at risk for said disease; b) determiningthe apoE genotype or phenotype of said patient; c) converting the dataobtained in step b) and determining the prognosis of said patient, saidprognosis including a prediction of whether the patient is a candidatefor a drug trial for the treatment of a neurological disease.
 13. Themethod of claim 12, wherein said method further comprises determiningthe BChE genotype or phenotype of said patient.
 14. The method of claim12 wherein said drug is from the group comprising aspirin,antithrombotics, ticlopidine, Ticlid™, cholinomimetics, tacrine,levodopa-carbidopa, Sinernet™, interferon β-1B, or Betaseron™.
 15. Themethod of claim 12 wherein said drug is tacrine.
 16. The method of claim12 wherein said patient is asymptomatic.
 17. A method of determining aprognosis of future risk of a disease for a mammal asymptomatic for saiddisease, said method comprising: a) determining the apoE genotype orphenotype of said mammal; b) converting the data obtained in step a)into a prognosis for said human, said prognosis including a predictionof a human's future disease risk, drug treatment efficacy for saiddisease, or treatment outcome.
 18. The method of claim 17, wherein saidmammal is a human.
 19. The method of claim 17, wherein the methodfurther comprises determining the BChE genotype or phenotype of saidmammal.
 20. The method of claim 17, wherein said method furthercomprises obtaining a patient profile of said mammal.
 21. The method ofclaim 20, wherein said patient profile includes a determination of saidmammal's sex.
 22. The method of claim 20, wherein said patient profileincludes the genotype of said mammal.
 23. The method of claim 20,wherein said patient profile includes the age of said mammal.
 24. A kitfor determining a prognosis, said kit including a means for convertingthe patient profile into a prognosis.
 25. The kit of claim 24, whereinsaid kit contains a means for performing the steps of said conversion.26. The kit of claim 24, wherein said kit contains a means for compilingthe data for said patient profile.
 27. The kit of claim 24, wherein saidkit contains a computer software program to perform the data analysis.